Demographic information for the clinical cohorts evaluated in this study.

<p>Definition of abbreviations: N = number of subjects providing DNA samples evaluated in this study; SD = standard deviation; FEV₁ = forced expiratory volume in 1 second (mL); LOCCS = Leukotriene Modifier Or Corticosteroid or Corticosteroid-Salmeterol Trial; LODO = Effectiveness of Low Dose Theophylline as Add On Therapy for the Treatment of Asthma; CLIC = Characterizing the Response to a LT Receptor Antagonist and Inhaled Corticosteroid trial; PACT = Pediatric Asthma Controller Trial.</p><p>Demographic information for the clinical cohorts evaluated in this study.</p

Improvement in lung function related to montelukast treatment, by rs6475448 genotype.

<p>The least-squares (LS) means (adjusted for study, race and gender) and 95% confidence intervals for ΔFEV₁ related to montelukast treatment were generated using R (<a href="http://cran.r-project.org/web/packages/lsmeans/lsmeans.pdf" target="_blank">http://cran.r-project.org/web/packages/lsmeans/lsmeans.pdf</a>), and plotted for each study (panels), by rs6475448 genotypes: homozygous reference (“GG”: LOCCS = 32; LODO = 38; CLIC = 25; PACT = 65), heterozygous (“GA”: LOCCS = 28; LODO = 21; CLIC = 30; PACT = 75) and homozygous variant (“AA”: LOCCS = 9; LODO = 5; CLIC = 5; PACT = 5).</p

Replicated^* GWAS SNPs.

<p>Definition of abbreviations: “SNP” = single nucleotide polymorphism; “Chr.” = chromosome (1–22); “Chr. Location.” = chromosomal position of listed SNP; “β” = effect size estimates (ΔFEV₁, (mL)) for the minor allele.</p><p><b>*</b>Table lists GWA results adjusted for baseline FEV₁, age, race and gender as covariates (additive genetic model), for the SNPs that met criteria for replication in all cohorts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129385#sec006" target="_blank">Methods</a>) and remained significant after correction for multiple testing. Minor allele frequencies for all SNPs in all cohorts is >5%.</p><p>^<b>‡</b>Combined P value for all cohorts.</p><p>Replicated^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129385#t002fn002" target="_blank">*</a>} GWAS SNPs.</p

Results of the discovery GWAS.

<p>Manhattan plots (<b>A</b> and <b>B</b>) contain −log P values (y-axis) associated with 8-week change in FEV₁ after montelukast treatment, for 532,264 genotyped SNPs organized by chromosome (x-axis), for LOCCS (<b>A</b>) and LODO (<b>B</b>). The threshold for genome-wide significance and suggestive genome-wide significance are indicated as blue and red lines, respectively, in the Manhattan plots. Q-Q plots (<b>C</b> and <b>D</b>) demonstrate the observed −log P values vs. expected −log P values, for SNPs from LOCCS (<b>C</b>) and LODO (<b>D</b>) populations. In all plots, individual SNPs are represented as filled circles.</p

Haplotypes with Copy Number and Single Nucleotide Polymorphisms in <em>CYP2A6</em> Locus Are Associated with Smoking Quantity in a Japanese Population

<div><p>Smoking is a major public health problem, but the genetic factors associated with smoking behaviors are not fully elucidated. Here, we have conducted an integrated genome-wide association study to identify common copy number polymorphisms (CNPs) and single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) in Japanese smokers ( = 17,158). Our analysis identified a common CNP with a strong effect on CPD (rs8102683; ) in the 19q13 region, encompassing the <em>CYP2A6</em> locus. After adjustment for the associated CNP, we found an additional associated SNP (rs11878604; ) located 30 kb downstream of the CYP2A6 gene. Imputation of the <em>CYP2A6</em> locus revealed that haplotypes underlying the CNP and the SNP corresponded to classical, functional alleles of CYP2A6 gene that regulate nicotine metabolism and explained 2% of the phenotypic variance of CPD (ANOVA -test ). These haplotypes were also associated with smoking-related diseases, including lung cancer, chronic obstructive pulmonary disease and arteriosclerosis obliterans.</p> </div

Manhattan plot showing the significance of association for all CNPs and SNPs in the genome-wide CPD analysis.

<p>The SNPs consist of both Illumina 610K chip and imputed HapMap SNPs. All CNPs and SNPs are plotted on the axis according to their positions on each chromosome against association with CPD on the axis (-value). SNPs and CNPs with -values are highlighted in green. A dark gray line shows the genome-wide significance level after Bonferroni's correction with a total of 2,312,503 SNPs and 4,256 CNPs.</p

Association of the haplotype between rs11878604 (SNP) and rs8102683 (CNP) for three dieseases.

<p>Association of the haplotype between rs11878604 (SNP) and rs8102683 (CNP) for three dieseases.</p

Frequency and relative effect size of haplotype between rs11878604 (SNP) and rs8102683 (CNP).

¶<p>Haplotypes consisting of alleles at rs11878604 [T/C] and haplotypic copy numbers at rs8102683 [0/1/2].</p>*<p>Haplotype frequencies and relative effect sizes are jointly evaluated using a haplotype-specific linear regression model as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044507#pone.0044507-Stram1" target="_blank">[27]</a>. All model parameters were estimated using a standard EM (expectation-maximization) algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044507#pone.0044507-McLachlan1" target="_blank">[28]</a>.</p>†<p>Variance explained by these six haplotypes and its significance was assessed in the standard ANOVA (analysis of variance) framework.</p

Role of Lung Function Genes in the Development of Asthma

<div><p>Although our previous GWAS failed to identify SNPs associated with pulmonary function at the level of genomewide significance, it did show that the heritability for FEV₁/FVC was 41.6% in a Japanese population, suggesting that the heritability of pulmonary function traits can be explained by the additive effects of multiple common SNPs. In addition, our previous study indicated that pulmonary function genes identified in previous GWASs in non-Japanese populations accounted for 4.3% to 12.0% of the entire estimated heritability of FEV₁/FVC in a Japanese population. Therefore, given that many loci with individual weak effects may contribute to asthma risk, in this study, we created a quantitative score of genetic load based on 16 SNPs implicated in lower lung function in both Japanese and non-Japanese populations. This genetic risk score (GRS) for lower FEV₁/FVC was consistently associated with the onset of asthma (<i>P</i> = 9.6 × 10⁻⁴) in 2 independent Japanese populations as well as with the onset of COPD (<i>P</i> = 0.042). Clustering of asthma patients based on GRS levels indicated that an increased GRS may be responsible for the development of a particular phenotype of asthma characterized by early onset, atopy, and severer airflow obstruction.</p></div

Signal plots before conditioning (a), after conditioning on rs8102683 (b) and after conditioning on rs8102683 and rs11878604 (c).

<p>SNPs and CNPs are plotted on the axis according to their positions on chromosome 19 (NCBI Build 37; hg19) against association with CPD on the left axis (-value). SNPs from the 1000 genomes imputation (phase I; 2011-11-23) are indicated with circles, SNPs genotyped on the Illumina 610K chip are indicated with triangles and CNPs genotyped with PlatinumCNV using the raw signal intensity data from the chip are indicated with diamonds. SNPs in the commonly deleted region (shown by the gray shaded area) are imputed as tri-allelic SNPs with deletions (see Materials and Methods for details). All SNP associations are assessed using allele dosages (the number difference between the A and B alleles; see Materials and Methods), and the CNP associations are assessed using the posterior mean copy number dosage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044507#pone.0044507-Kumasaka1" target="_blank">[8]</a>. Recombination rates (cM/Mb) across the region are shown by the purple line plotted against the right axis. The most significant variant of the SNP or CNP for each panel is pink, and the surrounding SNPs and CNPs are color-coded to reflect the strength of LD with the top variant according to the Pearson's values.</p